Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSfragment, with the tryptophan synthetase gene (trp1) of C. cinerea, into the ColE1 vector pUC9. The inserted gene allows the complementation of trp1auxotrophies and can be used as a selection marker. Several transformation experiments using this vector reveal a surprising phenomenon. Singletransformationwith solely pCc1001 gives only low numbers of transformants, whereas co-transformation with an additional plasmid yields about 2x moretransformants. To explore this phenomenon, the length of the trp1 harboring fragment was changed and the existing replicon was replaced by a modifiedColE1 replicon. All single-transformations resulted in the same observations. An alternative selection marker (pab1 encoding) and different relative vectorvectorconcentrations were tested in several co-transformation experiments. The obtained results lead to the conclusion that tryptophan feedbackinhibition might be responsible for the reduced transformation efficiencies in single-transformations of trp1 vectors. (1) Binninger et al. (1987). DNAmediatedtransformation of the basidiomycete Coprinus cinereus. EMBO J 6:835-840.471. The first promoter for conditional gene expression in Acremonium chrysogenum: iron starvation-inducible mir1 P . Fabio Gsaller 1 , Michael Blatzer 1 ,Beate Abt 1 , Markus Schrettl 2 , Herbert Lindner 3 , Hubertus Haas 1 . 1) Christian Doppler Laboratory for Fungal Biotechnology, Division of Molecular Biology,Medical University of Innsbruck, Austria; 2) Sandoz GmbH, Kundl, Austria; 3) Division of Clinical Biochemistry, Medical University of Innsbruck, Austria.The filamentous fungus Acremonium chrysogenum is of enormous biotechnological importance as it represents the natural producer of the beta-lactamantibiotic cephalosporin C. However, a limitation in genetic tools, e.g. promoters for conditional gene expression, impedes genetic engineering of thisfungus. Here we demonstrate that in A. chrysogenum iron starvation induces the production of the extracellular siderophores dimerumic acid, coprogen B,2-N-methylcoprogen B and dimethylcoprogen as well as expression of the putative siderophore transporter gene, mir1. Moreover, we show that thepromoter of mir1, mir1 P , is suitable for conditional expression of target genes in A. chrysogenum as shown by mir1 P -driven and iron starvation-inducedexpression of genes encoding green fluorescence protein and phleomycin resistance. The obtained iron-starvation dependent phleomycin resistanceindicates the potential use of this promoter for selection marker recycling. Together with easy scorable siderophore production, the co-regulation of mir1expression and siderophore production facilitates the optimization of the inducing conditions of this expression system. This work was funded by SandozGmbH (Kundl, Austria) and the Christian Doppler Society (Vienna, Austria).472. Mutagenic effect of high-LET ion beam irradiation in Neurospora crassa. Liqiu Ma 1* , Yusuke Kazama 2 , Tomoko Abe 2 , Shuuitsu Tanaka 1 , ShinHatakeyama 1 . 1) Regulation Biol, Saitama Univ, SAITAMA, Japan; 2) Radiation Biology Team, RIKEN, SAITAMA, Japan.Heavy ion beams cause great damages to cellular components particularly generating severe DNA damages, DNA double strand breaks (DSBs). Weexamined the biological effect and mutagenesis of irradiation of high-LET ion beam (Fe-ion) to DSB repair defect mutants in filamentous fungus Neuosporacrassa. Fe-ion beam ( 56 Fe 24+ : 90 MeV/u, LET=641 keV/mm) was irradiated to two DSB repair deficient mutants and wild-type strain. By lower doses (100 Gy), sensitivity to irradiation of the mus-52 strain (non-homologousend-joining deficient) is higher than that of the wild type, whilst lower than that of the mei-3 strain (homologous recombination deficient). Frequency offorward mutation occurred in the ad-3 loci was similar to previously examined C-ion beam irradiation, i.e. mei-3 > wild type > mus-52 strains. However,characteristic difference of mutation was observed as the scale of deletions; large deletions were frequently in the Fe-ion beam irradiated wild type strain,comparing to that 1 bp-deletions were mainly observed in the C-ion irradiation. Differences of mutagenesis and killing effect between the irradiation oftwo heavy ions, Fe-ion and C-ion, were discussed based on types of DNA damages.473. The Mad complex binds to light-regulated promoters in Phycomyces blakesleeanus. Alejandro Miralles-Duran, LM Corrochano. Genetica, Facultadde Biologia, University of Sevilla, Sevilla, Spain.The zygomycete Phycomyces blakesleeanus responses to light include phototropism of the fruiting body, activation of beta-carotene biosynthesis, andregulation of fruit body development. These photoresponses require the Mad complex, a protein complex composed of proteins MadA and MadB. Theseproteins are homologous of WC-1 and WC-2 from Neurospora crassa and presumably play a similar role in the regulation by light of gene expression.MadA and MadB have a zinc finger domain at the carboxyl end, and MadA has a LOV domain that should serve as the binding site for a flavinchromophore. In Phycomyces, the Mad complex should operate as a photoreceptor and transcription factor complex. The Phycomyces genome containstwo additional wc-1 homologs, wcoA and wcoB, and three additional wc-2 homologs, wctB, wctC, and wctD, but their function is unknown. We haveexpressed MadA and MadB in E. coli, and we have shown that these proteins bind the promoter of the light-regulated gene hspA by electrophoresismobility shift assays (EMSA). Protein binding to the hspA promoter was observed with each isolated protein or with the two proteins associated in theMad complex. The binding site to the hspA promoter will be identified by DNA footprinting analysis. We are performing similar assays with the otherPhycomyces Wc proteins and we hope that the results will help us to understand the role of the multiple Wc proteins in light-dependent gene regulation inPhycomyces.474. Down Regulation of sidB Gene by Use of RNA interference Technology in the Filamentous Fungi Aspergillus nidulans. S, Rezaie 1,2 , H, Eslami 1 , M.R.Khorramizadeh 1 , M.R. Pourmand 1 , M. Moazeni 2 . 1) Medical Biotechnology Dept, Tehran University of Medical Sciences, PhD; 2) Div. of Molecular Biology,Dept. of Medical Mycology and Parasitology, Tehran University of Medical Sciences, PhD.Background: RNA interference (RNAi) is a natural process by which short double-stranded RNA (siRNA) silences the expression of complementary targetRNAs by inducing RNA cleavage and subsequent reduction in protein expression levels. Introduction of the RNA interference machinery has guided theresearchers to discover novel methodologies for knocking down essential vital factor or virulence factor genes in the microorganisms such as fungi. Infilamentous fungi, Aspergillus nidulans, the gene sidB plays essential role in septation, conidiation and vegetative hyphal growth. In the present study, webenefited from the RNA interference strategy for down-regulating of a vital gene in the fungus Aspergillus nidulans. Materials and Methods: The 21-nucleotide siRNA was designed on the basis of the cDNA sequence of the sidB gene of A. nidulans. Transfection was performed via uptaking siRNAs frommedium by germinated spores. After 18 hours of incubation, total RNA was extracted and quantitative changes in expression of the sidB gene wereanalyzed by measuring the cognate sidB mRNA level by use of a quantitative real-time RT-PCR assay. Results: In the presence of 25 nM of siRNA, asignificant inhibition in germ tube elongation was observed compared with positive control samples (21 VS 42 mM). In addition, at the concentration of 25nM , a considerable decrease in sidB gene expression was revealed. Conclusion: Usage of RNA interference as a kind of post-transcriptional gene silencingmethods is a promising approach for designing new antifungal agents and discovering new drug delivery systems.475. SmallRNA mediated meiotic silencing of a transposable element in Neurospora crassa. Yizhou Wang, Jason E. Stajich. Plant Pathology &Microbiology, Univ. of CA, Riverside, Riverside, CA.Meiotic silencing of unpaired DNA plays an important role in protecting the genome integrity of Neurospora crassa. It is thought to fight against theinvasion of virus and endogenous transposable elements. Our previous work has shown that a 10 KB MULE (mutator-like element)-related DNA27th Fungal Genetics Conference | 237